Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

SV40 virus infection is able to induce tumours in newborn hamsters and to transform a wide range of eukaryotic cells in in vitro culture. This is achieved by integration of the viral DNA into the host cell DNA and expression of the virus-encoded Large T-antigen. The expression of Large T, a 708 amino acid phosphoprotein, is required both to induce and maintain the transformed state. The Large T protein initiates viral DNA synthesis and regulates viral transcription, apparently by binding in a specific manner to viral DNA sequences at and near the viral origin of replication. SV40 Large T also affects cellular DNA synthesis and transcription and this may account for its oncogenic activity. A novel immunochemical procedure has permitted the isolation of cellular DNA sequences occupied by SV40 Large T in the chromatin of SV40 transformed cells. Some of the cellular sequences contain high affinity binding sites for SV40 Large T, and hybridize to messenger RNAs expressed in SV40 transformed but not in normal cells. A second type of cellular target for Large T is the cell coded p53 protein that it binds to and stabilizes. A range of monoclonal antibodies to p53 has been isolated and characterized. They demonstrate that p53 is in the cytoplasm of normal cells but is located in the nucleus of transformed cells. One of the antibodies recognizes an epitope on p53 that is stabilized or induced by binding to Large T. Further studies on the T-p53 protein complex have been facilitated by constructing bacterial plasmids that direct the synthesis of substantial quantities of Large T-beta-galactosidase and p53-beta-galactosidase fusion proteins in bacteria. The results are discussed in the context of our current knowledge of oncogene action.
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PMID:Cellular targets for SV40 large T-antigen. 241 84

p53 mRNA and proteins were examined in a variety of human transformed cells and in normal human foreskin fibroblast cells. Both the steady-state and translatable levels of p53 mRNA were the same in normal and transformed human cells. In vitro synthesized p53, programmed by mRNA from normal and transformed human cells, revealed that there was heterogeneity in the primary structure of p53 from these cells. Pulse labeling of cells and immunoprecipitation analysis with a panel of human reactive anti-p53 antibodies demonstrated that the types of p53 synthesized in vitro corresponded to the types made in vivo from SV80 and COLO 320 cells. No p53 was detectable by similar pulse-labeling analysis of HeLa and normal foreskin fibroblast cells. Since it was necessary to use anti-p53 sera from cancer patients to carry out much of the immunoprecipitation analysis in this study we therefore further characterised these sera to determine if they reacted with one or more than one epitope. p53-beta-galactosidase fusion proteins were synthesized in Escherichia coli and used to analyse the anti-p53 antibodies produced by cancer patients. We demonstrate that the antisera contain antibodies directed against epitopes in both the N-terminal and C-terminal regions of the p53 molecule.
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PMID:Analysis of human p53 proteins and mRNA levels in normal and transformed cells. 241 31

The isolation and construction of a complete human p53 cDNA and subsequent expression in monkey cells is described. A set of new anti-(human p53) monoclonal antibodies has also been obtained and used to show the expression of the human p53 cDNA in cos-l cells. These antibodies enable the specific detection of human p53, which is synthesised in the presence of p53 from other species. Fusion proteins of p53 with beta-galactosidase were used firstly as antigen and secondly, in conjunction with competition assays, to localise the determinants recognized by the antibodies. At least two previously unrecognized epitopes are involved and two of the antibodies are human-p53-specific. The epitopes are denaturation-resistant and the antibodies are, therefore, valuable for immunoblotting as well as immunoprecipitation and enzyme-linked immunoassay. Transfection of plasmids containing complete human p53 cDNA into monkey (cos-l) cells cause expression of human p53 recognized by the monoclonal antibodies. Control plasmids did not induce immunoreactive protein.
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PMID:Isolation of human-p53-specific monoclonal antibodies and their use in the studies of human p53 expression. 242 16

We developed a rapid assay for identifying growth-arrest genes to facilitate studies of cell cycle regulation. A7r5 vascular smooth muscle cells were transiently transfected with two plasmids: (i) a pMSV beta Gal reporter construct expressing beta-galactosidase (beta-gal) under transcriptional control of the murine sarcoma virus long terminal repeat; and (ii) a eukaryotic expression vector driving transcription of a potential growth inhibitory c-DNA under control of the cytomegalovirus promoter/enhancer. Twenty-four hours after transfection, cellular DNA was labeled for an additional 24 h with 5-bromo-2-deoxyuridine (BrdU) to label cellular DNA. After fixation, transfected cells were identified by histochemical staining with a beta-gal substrate, 6-chloro-3-indolyl-beta-D-galactopyranoside (i.e., Red-Gal). Transfected cells (beta-gal-positive) that traversed S phase (i.e., DNA synthesis) were quantified by indirect immunocytochemical staining for BrdU. Since autoradiography was not required to score for DNA synthesis, the length of experiments was much shorter than previously described growth-arrest assays performed with transiently transfected cells. Experiments with two growth-arrest genes, p53 and the p21 cyclin-dependent kinase inhibitor, demonstrated the utility of this assay.
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PMID:Rapid characterization of growth-arrest genes in transient transfection assays. 754 21

Mutations of the p53 tumor suppressor gene are the most common molecular genetic abnormality to be described in ovarian cancer. To determine the feasibility of mutant p53 as a molecular target for gene therapy in ovarian cancer, we constructed an adenovirus vector containing the wild-type p53 gene. The ability of this adenovirus construct (Ad-CMV-p53) to express p53 protein was examined by Western blot analysis in the H358 lung cancer cell line, which has a homozygous deletion of the p53 gene. The ability of the adenovirus vector system to infect ovarian cancer cells was tested using an adenovirus containing the beta-galactosidase reporter gene under the control of the CMV promoter (Ad-CMV-beta gal). The ovarian cancer cell line 2774, which contains an Arg273His p53 mutation, was infected with Ad-CMV-beta gal, and the infected cells were assayed for beta-galactosidase activity after 24 hr. To test the ability of wild-type p53 to inhibit cell growth, the 2774 cell line was infected with Ad-CMV-p53 or Ad-CMV-beta gal, and the effect of these agents on the growth of 2774 cells was determined using an in vitro growth inhibition assay. Western blot analysis of lysates from H358 cells infected with Ad-CMV-p53 showed expression of wild-type p53 protein. When 2774 cells were infected with Ad-CMV-beta gal at a multiplicity of infection (m.o.i.) of 10 PFU/cell, > 90% of cells showed beta-galactosidase activity, demonstrating that these cells are capable of efficient infection by the adenovirus vector. Growth of 2774 cells infected with Ad-CMV-p53 was inhibited by > 90% compared to noninfected cells. The ability of the adenovirus vector to mediate high-level expression of infected genes and the inhibitory effect of Ad-CMV-p53 on the 2774 cell line suggests that the Ad-CMV-p53 could be further developed into a therapeutic agent for ovarian cancer.
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PMID:Adenovirus-based p53 gene therapy in ovarian cancer. 759 Apr 66

Studies on the molecular basis of human breast cancer have demonstrated that mutational inactivation of the p53 tumor suppressor gene may be an essential step in the development of this cancer. We and others have previously shown that transfer of the wild-type p53 gene into cultured breast cancer cells reduced their malignant potential. We report here on a p53 gene transfer protocol based on a replication-incompetent retrovirus to efficiently inhibit tumor formation of cancer cells with endogenous mutant p53. The susceptibility of the cells to retroviral infection was determined with LZRNL transducing the lacZ reporter gene. A multiplicity of infection (moi) of 2 resulted in 90% of the exposed cell population in cytochemically detectable beta-galactosidase activity. Using the p53 vector Lhp53RNL with a moi of 2 was sufficient to completely supress tumor formation by the highly tumorigenic MDAMB231 breast cancer cells carrying a point missense mutation in codon 280. Even after 12 weeks, no vital tumors were histologically detectable. For comparison, established protocols were used to infect MDAMB231 cells with low moi with the p53 virus. Clones were expanded in G418-selective media for few weeks, pooled and injected into nude mice. Tumor formation occurred already after 1 week from G418-selected cells. Long-term expression of the p53 transgene was more stable in retrovirally bulk-infected and nonselected cells resulting in an efficient suppression of tumor formation. This approach may facilitate future studies on other growth suppressive genes that potentially qualify for in vivo gene therapy.
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PMID:p53 trans-dominantly suppresses tumor formation of human breast cancer cells mediated by retroviral bulk infection without marker gene selection: an expeditious in vitro protocol with implications towards gene therapy. 759 Jul 73

We report on an in vivo delivery system that attenuates the growth, in nude mice, of a malignant human breast cancer cell line containing a p53 mutation. Nude mice, inoculated with breast carcinoma cells, were injected every 10-12 days with a liposome-p53 complex via the tail vein. A significant reduction of greater than 60% in primary tumor volume was observed as compared to the control groups. Furthermore, when individual growth patterns of the tumors were assessed, we found that primary tumor size regressed in the majority of p53-treated animals (8/15), whereas only one tumor in the control groups (1/22) regressed. The eight tumors that regressed with the liposome-p53 complex showed no evidence of relapse for 1 month after the cessation of treatment. We also determined that the administration of the liposome-p53 complex reduced the incidence of metastases. The MDA-MB-435 tumor cells, transduced with the lacZ gene, facilitated quantitation of beta-galactosidase activity and tumor burden in the lungs. The number of metastatic cells in the lung was significantly lower in the p53-treated group (0.53 +/- 0.43 x 10(6), p < 0.01) than in either the vector-treated (8.1 +/- 3.7 x 10(6)) or untreated control groups (15.8 +/- 5.9 x 10(6)). Thus, systemic administration of the liposome-p53 complex reduced not only the size of the primary tumors but, more importantly, prevented the relapse and metastases of these tumors.
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PMID:Systemic gene therapy with p53 reduces growth and metastases of a malignant human breast cancer in nude mice. 761 97

Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.
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PMID:A proteolytic pathway that recognizes ubiquitin as a degradation signal. 761 50

Adenoviral vectors have recently been shown to effectively deliver genes into a variety of tissues. Since these vectors have some advantages over the more extensively investigated retroviruses, we studied the effect of two replication-defective adenovectors bearing human wild type tumor suppressor gene p53 (Adp53) and Escherichia coli beta-galactosidase gene (AdLacZ) on 9L glioma cells. Successful in vitro gene transfer was shown by DNA polymerase chain reaction (PCR), and expression was confirmed by reverse transcriptase RNA PCR and Western blot analyses. Transduction of 9L cells with the Adp53 inhibited cell growth and induced phenotypic changes consistent with cell death at low titers, while AdLacZ caused cytopathic changes only at high titers. Stereotactic injection of AdLacZ (10(7) plaque forming units) into tumor bed stained 25 to 30% of tumor cells at the site of vector delivery. Injection of Adp53 (10(7) plaque forming units), but not AdLacZ (controls), into established 4-day old 9L glioma brain tumors decreased tumor volume by 40% after 14 days. As a step toward gene therapy of brain tumors using replication-defective adenoviruses, these data support the use of tumor suppressor gene transfer for in vivo treatment of whole animal brain tumor models.
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PMID:Adenovirus-mediated p53 gene delivery inhibits 9L glioma growth in rats. 764 77

Two steps of gene targeting were used to replace the p53 gene with the E. coli beta-galactosidase (lacZ) gene in mouse embryonic stem (ES) cells. The first targeting vector consisted of neo and herpes simplex virus thymidine kinase (HSV-tk) genes as a neo-tk cassette in the middle of the targeting vector. At the first targeting, the homologous recombinants became G418 resistant and ganciclovir (GANC) sensitive and were selected by G418 alone. At the second targeting, homologous recombination reciprocally exchanged the neo-tk casette in the ES cell chromosome with the lacZ fragment in the second targeting vector and thus made the ES cells GANC resistant. We obtained two ES cell clones, in which the p53 gene for both had been replaced with a totally non-homologous sequence of the lacZ gene. The germ-line transmission of the manipulated ES cells also demonstrated that the entire procedure had no detrimental effects on ES cells at all.
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PMID:Gene replacement of the p53 gene with the lacZ gene in mouse embryonic stem cells and mice by using two steps of homologous recombination. 804 55


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